Anellovirus genome quantification as a biomarker of immune suppression

ABSTRACT

The present invention relates to the use of the measure of anelloviral load for the determination of immunosuppression. More precisely, the present invention provides a method for characterizing the immunosuppressed or non-immunosuppressed status of a subject, comprising the steps of determining the anelloviral load from a biological sample of the said subject, and determining from the said comparison the immunosuppressed or non-immunosuppressed status. The determination of the immunosuppressed status of the subject can then be used to design or adapt a therapeutic treatment.

The present invention relates to the use of the measure of anelloviralload for the determination of immunosuppression. More specifically, theinvention relates to a method for the diagnosis of immunosuppression ina subject based on anellovirus viral load.

The immune system defends an organism against aggressions such aspathogen infection, cellular transformation, and physical or chemicaldamage. When the immune system is less active than normal,immunodeficiency or immunosuppression occurs, resulting inlife-threatening infections or cancer. Immunosuppression is a conditionin which the immune system's ability to fight diseases, for exampleinfectious diseases or cancer, is compromised or entirely absent.Immunosuppression takes various forms, and may affect either the innateor the adaptive immune system, or both, depending of the source of thedeficiency. It usually results in recurring or life-threateninginfections.

Immunosuppression can either be the result of diseases, or be producedby pharmaceuticals or an infection, resulting in an increasedsusceptibility to secondary infections by pathogens such as bacteria andviruses.

Many diseases are thus characterized by the development of progressiveimmunosuppression in the patient. The presence of an impaired immuneresponse in patients with malignancies (e.g. leukemia, lymphoma,multiple myeloma) is well documented. Progressive immunosuppression hasalso been observed in certain chronic infection such as AIDS, sepsis,leprosy, cytomegalovirus infections, malaria, lupus, and the like.Immunodeficiency is also a potential adverse effect of many therapeutictreatments (radiotherapy or chemotherapy for example). In such asituation of non-deliberate immunosuppression, patients are usuallytreated with immunostimulants (e.g. cytokines), in order to boost thepatient's immune system. However, immunostimulants lack specificity, inthat they activate the immune system in general. If not administeredcautiously, they may trigger an overactivation of the immune system,resulting in poor tolerance

Alternatively, immunosuppression may result from deliberate intent toweaken the immune system. In general, deliberately inducedimmunosuppression is performed by administration of immunosuppressivedrugs, in order to prevent the body from rejecting an organ transplantor for the treatment of auto-immune diseases. Immunosuppressivetreatments, however, when inappropriate or inadequate, may lead to anover-immunosuppression state where the patient is extremely vulnerableto infections. Indeed, opportunistic infections and malignancies remaina significant cause of death after transplantation and are obviousconsequences of over-immunosuppression.

Because of the great diversity of causes, and because each of thosecauses may affect the immune system in a different aspect, differentdiagnosis for immunosuppression have been developed. Some nonspecificand pathogen-specific measures of cell-mediated immune function areavailable (Fishman et al. N Engl J Med. 2007, Fishman et al. LiverTranspl. 2011). Cell mediated immune function assays include lymphocytesubset analysis, particularly CD4⁺ T cell numbering or measure of theCD4+/CD8+ T cell ratio, neutrophil function assay, NK activation assay,lymphocyte proliferation assay (Hutchinson et al. Nephrol DialTransplant 2003).

Those assays however are not sufficiently sensitive to detect slightchanges in the immune system. Additionally, each of those assays focuson assaying the integrity of a specific pathway or mechanism of theimmune system. None of them is based on assaying the end result, whichis the capability of the immune system to respond to or controlinfections.

Currently there is a no universal method for the diagnosis ofimmunosuppression that could be used universally, that is, for anysuspected cause of immunosuppression. There is thus a ongoing need for arapid, reliable and non-invasive test assessing precisely the immunestatus of a patient. Especially, a diagnosis method that would allow forevaluation of the capacity of the immune system to respond to infectionscould be used to fine-tune immunosuppressive treatments to the properneeds of the patients, and avoid over-immunosuppression.

The inventors have found that the anelloviral load is a reliable markerof immune status and can thus be used for the diagnosis ofimmunosuppression.

DETAILED DESCRIPTION

Anelloviruses (ANV) are viruses that infect more than 90% individuals.Mixed infections with several strains and ANV species are frequent, andmost subjects host at least one of them, but no pathological consequencehas been attributed to ANV infection. In particular, no clearcorrelation between the anelloviral load and the immune state of thesubject has been identified in the prior art. Patients onimmunosuppressive treatment generally showed an increase in the load ofspecific viral strains (e.g. TLMV and TTV). However, theinter-individual variations were such that the only relevant informationcould be obtained from examining the changes in the viral load of eachindividual. In particular, no conclusion could be drawn from thecomparison of groups of patients, treated or not treated. Thus themethods of the prior art did not enable the determination of theimmunosuppressive status of a subject and the design of a specifictreatment thereof (Moen et al., J Med Virol, 70(1): 177-182, 2003; Moenet al., AIDS, 16(12): 1679-1682, 2002; Christensen et al., J Infect Dis,181: 1796-1799, 2000; Shibayama et al., AIDS, 15: 563-570, 2001;Touinssi et al., J Clin Virol, 21: 135-141, 2001).

In contrast, the present inventors have surprisingly found that theanelloviral load can be used as a marker for immunosuppression. Whereasprevious studies were based on PCR, and thus were highly dependent uponprimers design, resulting in missing variants of this highly variablevirus (Moen et al., J Virol Methods,104(1): 59-67, 2002), the presentinventors used High Throughput Sequencing (HTS). This technique led tothe identification of a broad and unbiased range of ANV sequences,enabling the present inventors to demonstrate the existence of acorrelation between the anelloviral load and immunosuppression with ahigh degree of confidence. In particular, the inventors have found thatimmunosuppressed subjects have a higher anelloviral load by comparisonwith healthy subjects.

The measure of the global load of the viruses from the family ofanelloviruses can thus be used as a marker of the immune state of thesubject. More precisely, according to the invention, a high anelloviralload in a subject indicates that the said subject is immunosuppressed.

Thus, in a first aspect, the invention relates to a method forcharacterizing the immunosuppressed or non-immunosuppressed status of asubject, comprising the steps of:

-   a) determining the anelloviral load from a biological sample of the    said subject, and-   b) assessing from the determination of step a) the immunosuppressed    or non-immunosuppressed status.

The term “immunosuppression” (or “immunodepression” or“immunodeficiency”), as used herein, refers to the reduction orsuppression of the immune system function, i.e. immunosuppressiongenerally denotes a state when a subject's specific and/or non-specificimmune system function is reduced or absent. The whole immune responsemay be depressed, or a particular population of immunologically activelymphocytes may be selectively affected. In some cases, the effect maybe preferentially on T cells rather than B cells. If B cells areaffected, it may be on a specific subclass of antibody-producing cells.Antigen-specific immunosuppression may be the result of deletion orsuppression of a particular clone of antigen-specific cells, or theresult of enhanced regulation of the immune response by antigen-specificsuppressor cells. It can also be the result of increased production ofantiidiotypic antibody.

Immunosuppression may result from certain diseases such as AIDS orlymphoma or from certain drugs such as some of those used to treatcancer. Immunosuppression may also be deliberately induced with drugs,as in preparation for bone marrow or other organ transplantation toprevent the rejection of the transplant. Thus immunosuppressionaccording to the invention may be from any origin such as, for example,but not limited to, immunosuppressive treatment, immunosuppressive sideeffects of drugs or therapy including radiotherapy, inheritedimmunosuppressive genetic traits or diseases, acquired immunosuppressivediseases such as AIDs, cancers such as leukemia or lymphoma.

By “immunosuppressed status” it is herein referred to a condition wherethe immune system function of a subject is reduced or absent. Thus,according to the invention the terms “immunosuppressed”,“immunodepressed” or “immunocompromised” are all deemed to carry thesame meaning. In a particular embodiment, the “immunosuppressed status”of the subject means the ability of the subject to control viralinfection, that is to say, the ability of the subject to prevent viralamplification from said viral infection.

It is difficult to precisely assess the immunosuppressed status of asubject with the methods of the prior art. This may lead to situationswhere too high a dose of an immunosuppressive treatment is given to apatient in need thereof. It is also possible that a dose not high enoughof an immunostimulating treatment is administered to a patient, becausethe immunosuppressed status of the said patient was not correctlydetermined. The consequences for the patient's health are potentiallyserious in either situation. For example, immunosuppressive treatments,when inappropriate or inadequate, may lead to an over-immunosuppressionstate, which leaves the patient highly susceptible to infections. On theother hand, it is important to be capable of identifying reliablychronically immunosuppressed patients, so as to provide them with themost adequate treatment.

The method of the invention allows the precise determination of theimmunosuppressed status of the subject, enabling a specific treatment tobe tailored to the needs of the patient. The prior determination of theimmunosuppressed status of the patient with the method of the inventionthus lead to a treatment safer than the treatments designed on the basisof the methods of the prior art.

Thus the present invention also relates to a method for designing animmunomodulation treatment for a subject, said method comprising:

-   a) determining from a biological sample of a subject the anelloviral    load, and-   b) assessing from the determination of step a) the immunosuppressed    or non-immunosuppressed status, and-   c) designing the immunomodulation treatment according to said    immunosuppressed or non-immunosuppressed status assessed in step b)

The invention is also drawn to a method of treatment of a conditionassociated with immunodeficiency. As used herein; “conditions associatedwith immunodeficiency” or “immunodeficiency disorders” refer to adiverse group of conditions characterized primarily by an increasedsusceptibility to various opportunistic infections with consequentsevere acute, recurrent or chronic disease. In a first embodiment, thisincreased susceptibility to infection results from immunosuppression dueto one or more defects in the immune system. Immunosuppression in thiscase is non-deliberate. Immunodeficiency disorders encompass, withoutlimitation, “immunodeficiency syndromes” wherein all features are theresult of the immune defect, and “syndromes with immunodeficiency”,wherein some, even prominent features cannot be explained by the immunedefect. By means of example and not limitation, diseases and conditionsassociated with immunodeficiency or immunosuppression comprise: humanimmunodeficiency virus (HIV) infection and acquired immune deficiencysyndrome (AIDS), hypogammaglobulinemia, hematologic cancers such asleukaemia and lymphoma, lymphocytopenia (lymphopenia) of any origin,lupus erythematosus, cachexia, opioids abuse, mastocytosis, rheumaticfever, trypanosomiasis, alcohol abuse.

The group of immunodeficiency disorders also encompasses diseases andconditions associated with immunosuppression arising from an artificial,usually controlled diminution or prevention of a subject's immuneresponse. Immunosuppression in subjects is thus deliberately induced. Itmay be caused by immunosuppressive treatment, or it may occur as a sideeffect of a therapy of other indications (e.g., side effect of cancerchemotherapy). These latter conditions include such conditions as totalbone marrow ablation, bone marrow transplantation, organtransplantation, treatment with immunosuppressive drugs such as interalia tacrolimus, cyclosporine, methotrexate, mycophenolate,azathioprine, interferons, and immunoglobulins such anti-CD20 andanti-CD3; and treatments with: chemotherapy agents, corticosteroids,anti-TNF drugs, radiation.

Thus the present invention also relates to a method for treating acondition associated with immunodeficiency in a subject, said methodcomprising:

-   a) determining the immunosuppressed or non-immunosuppressed status    of the said subject according to the methods of the invention, and-   b) adapting an immunomodulation treatment to the said subject.

The present invention thus provides an immunomodulation treatment foruse in treating a condition associated with immunodeficiency in asubject, wherein the use comprises the steps of:

-   a) the immunosuppressed or non-immunosuppressed status of the said    subject is determined according to the methods of the invention, and-   b) the said immunomodulation treatment is adapted to the said    subject.

In other words, the invention relates to the use of an immunomodulationtreatment in the preparation of a medicament for treating a conditionassociated with immunodeficiency in a subject, wherein:

-   a) the immunosuppressed or non-immunosuppressed status of the said    subject is determined according to the methods of the invention, and-   b) the said immunomodulation treatment is adapted to the said    subject.

By “immunomodulation treatment”, it is herein referred to any treatmentintended to induce, enhance, inhibit or suppress an immune function.According to a preferred embodiment, an immunomodulation treatment is animmunosuppressive treatment. According to another preferred embodiment,an immunomodulation treatment is an immunostimulating treatment.

By “immunosuppressive treatment” it is herein referred to any treatmentintended to inhibit or suppress an immune function of a subject thatwould be adverse to a desired clinical outcome. Immunosuppressivetreatments include for example treatments that are intended to induceimmune function deficiency in a subject in order to treat the saidsubject with a transplant of cells or of an organ. They also include forexample treatments that induce immune function deficiency as a sideeffect, such as chemotherapy or radiotherapy. Immunosuppressivetreatments usually include e.g. glucocorticoids, antiproliferative andantimetabolic drugs (rapamycin, everolimus, mycophenolate mofetil,mycophenolic acid), calcineurin inhibitors (cyclosporine, FK506,voclosporin), S1P-R agonists (FTY720), malononitrilamides (FK778), andantibodies (e.g. antithymocyte globulin) including monoclonal antibodies(e.g. muromonab-CD3, daclizumab, basiliximab, rituximab, alemtuzumab,infliximab, adalimumab, efalizumab).

An “immunostimulating treatment”, according to the present invention, isany type of treatment intended to induce or enhance immune function.Immunostimulating treatments include treatments that stimulate specificimmune response, treatments that stimulate non-specific immune responseand treatments that stimulate both specific and non-specific immuneresponses. Such immunostimulating treatments are usually given to treatconditions associated with non-deliberate immunosuppression.Immunostimulating treatments that stimulate the immune response havebeen described in the literature, ranging from small synthetic molecules(poly I:C, levamisole, inosine pranobex) to living microorganisms(Corynebacterium parvum), and including complex mixtures of bacterialcomponents with mineral oils (Freund's adjuvant) or inorganic salts(aluminum and magnesium hydroxide/phosphate) and, more recently,recombinant proteins modulating immunity (e.g. cytokines, antibodiesagainst cellular receptors). According to the present inventionanti-viral treatments are also considered immunostimulating treatments.Anti-viral treatments include for example oseltamivir (Tamiflu),zanamivir (Relenza), interferon, which inhibit viral synthesis ininfected cells, particularly alpha-interferon, used in the treatment ofhepatitis B and C.

Thus, in one embodiment of the method of the invention, theimmunomodulation treatment is an immunosuppressive treatment. In anotherembodiment of the method of the invention, the immunomodulationtreatment is an immunostimulating treatment.

The said adaptation of the immunomodulation treatment may be either areduction or suppression of the said immunomodulation treatment or thecontinuation of the said immunomodulation treatment at the same or anincreased dose. The skilled person will appreciate that the treatmentwill be continued if the desired effect on the subject's immune systemis not achieved. For example, when the treatment is administered forstimulating the immune system function in order to compensate for adeficit thereof, the treatment is continued if the patient shows animmunosuppressed phenotype. Likewise, an immunosuppressive treatmentwill be continued if the subject displays a non-immunosuppressed status.On the other hand, the treatment will be reduced or suppressed if thedesired effect on the subject's immune system has been attained. This isthe case for example when the treatment seeks to obtainimmunosuppression in order to perform e.g. organ transplantation, whilethe subject shows an immunosuppression status.

In a preferred embodiment, the condition associated withimmunodeficiency is transplant rejection.

Thus, the invention relates to a method for treating or preventingtransplant rejection in a transplanted subject, said method comprising:

-   a) determining the immunosuppressed or non-immunosuppressed status    of the said transplanted subject according to the methods of the    invention, and-   b) adapting an immunosuppressive treatment to the said transplanted    subject.

The present invention thus provides an immunosuppressive treatment foruse in treating or preventing transplant rejection in a transplantedsubject, wherein the said use comprises the steps of:

-   a) determining the immunosuppressed or non-immunosuppressed status    of the said transplanted subject according to the methods of the    invention, and-   b) adapting the said immunosuppressive treatment to the said    transplanted subject.

In other words, the invention relates to the use of an immunosuppressivetreatment in the preparation of a medicament for treating or preventingtransplant rejection in a transplanted subject, wherein:

-   a) the immunosuppressed or non-immunosuppressed status of the    transplanted said subject is determined according to the methods of    the invention, and-   b) the said immunosuppressive treatment is adapted to the said    transplanted subject.

In another preferred embodiment, the condition associated withimmunodeficiency is an infection.

Thus this embodiment relates to a method of treating an infection in aninfected subject, said method comprising:

-   a) determining the immunosuppressed or non-immunosuppressed status    of the said infected subject according to the methods of the    invention, and-   b) adapting an immunostimulating treatment to the said infected    subject.

The present invention thus provides an immunostimulating treatment foruse in treating an infection in an infected subject, wherein the usecomprises the steps of:

-   a) determining the immunosuppressed or non-immunosuppressed status    of the said transplanted subject according to the methods of the    invention, and-   b) adapting the said immunostimulating treatment to the said    subject.

In other words, the invention relates to the use of an immunostimulatingtreatment in the preparation of a medicament for treating an infectionin an infected subject, wherein:

-   a) the immunosuppressed or non-immunosuppressed status of the    transplanted said subject is determined according to the methods of    the invention, and-   b) the said immunostimulating treatment is adapted to the said    transplanted subject.

An anellovirus according to the invention is a non-enveloped virus, witha small circular single-stranded DNA genome which is replicated throughdouble-stranded intermediates, and which may contain up to 4 openreading frames: ORF1, ORF2, ORF3 and ORF4. Open reading frames (ORF) 1(long) and 2 (short) are partially overlapping. ORF3 and ORF4 aresmaller. Anelloviruses are subgrouped into torque teno virus (TTV),torque teno mini virus (TTMV), and torque teno midi virus (TTMDV), withknown hosts including humans, non-human primates and domestic animals(Biagini et al., J Gen Virol, 88: 2696-2701, 2007; Hino

Miyata, Rev Med Virol, 17: 45-57, 2007; Leary et al., J Gen Virol, 80:2115-2120., 1999).

By “anellovirus”, it is herein referred to any virus belonging to theAnelloviridae family of viruses, including, but not limited to, theTorque Teno viruses (TTVs), the Torque Teno midiviruses (TTMDVs), andthe Torque Teno miniviruses, also formerly known as the Torque Teno-likeminiviruses (TTMVs). The prototype strain of Torque Teno Virus (TTV-1a)has a genome size of 3853 nucleotides. The prototype strain of TorqueTeno Minivirus, formerly known as TTV-like minivirus (TTMV-NLC030), hasa genome size of 2915 nucleotides. Finally, the Torque Teno Midivirushas been described, with a genome of 3242 nucleotides for the prototypestrain (TTMDV-MD1-073). Anelloviruses are highly variable in sequence.For example, nucleotide sequences of full-length genomes of TTV can varyby 40%, and those of TTMDV by 33%.

The “viral load” according to the invention is the number of nucleicacid sequences of a virus present in a biological sample. The viral loadreflects the severity of a viral infection. Preferably, the viral loadrefers to the proportion of a nucleic acid sequences in a biologicalsample which belong to the said virus. More preferably, the viral loadrepresents the number of copies of the said virus in a biologicalsample.

The viral load can for example be determined by estimating the amount ofthe virus in a biological sample from a subject.

As used herein, the term “subject” refers to a vertebrate, preferably amammal, and most preferably a human.

By “biological sample”, it is herein referred to any sample that istaken from a subject, which includes but is not limited to, for example,blood, serum, plasma, sputum, urine, stool, skin, cerebrospinal fluid,saliva, gastric secretions, semen, seminal fluid, tears, spinal tissueor fluid, cerebral fluid, trigeminal ganglion sample, a sacral ganglionsample, adipose tissue, lymphoid tissue, placental tissue, upperreproductive tract tissue, gastrointestinal tract tissue, male genitaltissue and fetal central nervous system tissue. Preferably, thebiological sample is blood or is derived from blood, such as plasma orserum.

According to the invention, the anelloviral load in a subject means theviral load of any virus of the Anelloviridae family hosted by saidsubject. Thus determining the anelloviral load in a subject according tothe invention comprises estimating the number of sequences of any virusof the Anelloviridae family in a biological sample from the saidsubject. In particular, there is no selection, according to theinvention, of specific anellovirus species to be measured in the saidbiological sample. Preferably, determining the anelloviral loadcomprises determining the amount of active and/or inactive viral copies.It comprises determining the amount of circulating, integrated or latentviral copies. More preferably, the anelloviral load corresponds tocirculating copies of anellovirus.

The levels of anellovirus may be determined by measuring levels ofanellovirus DNA, anellovirus RNA, or anellovirus proteins. The methodaccording to the invention may thus comprise another preliminary step,between the taking of the sample from the patient and step a) as definedabove, corresponding to the transformation of the biological sample intoa double-stranded DNA (dsDNA) sample, or into an mRNA (or correspondingcDNA) sample, or into a protein sample, which is then ready to use forin vitro detection of anellovirus in step a). The said dsDNA maycorrespond either to the whole anellovirus genome or only to a portionof it. Once a ready-to-use dsDNA, mRNA (or corresponding cDNA) orprotein sample is available, the detection of the anellovirus may beperformed, depending on the type of transformation and the availableready-to-use sample, either at the genomic DNA (i.e. based on thepresence of at least one sequence consisting of at least a part of theanellovirus genome), mRNA (i.e. based on the mRNA content of the sample)or at the protein level (i.e. based on the protein content of thesample).

Preferably, the levels of anellovirus are determined by measuring levelsof anellovirus DNA.

Methods for detecting a nucleic acid in a biological sample includeinter alia hybridization with a labelled probe, amplification, includingPCR amplification, sequencing, and all other methods known to the personof skills in the art. The amount of nucleic acid transcripts can bemeasured by any technology known by the skilled person. In particular,the measure may be carried out directly on an extracted messenger RNA(mRNA) sample, or on retrotranscribed complementary DNA (cDNA) preparedfrom extracted mRNA by technologies well-known in the art. From the mRNAor cDNA sample, the amount of nucleic acid transcripts may be measuredusing any technology known by a person skilled in the art, includingnucleic microarrays, quantitative PCR, and hybridization with a labelledprobe.

In a first embodiment of the invention, the levels of anellovirus DNAare measured by sequencing. As used herein, the term “sequencing” isused in a broad sense and refers to any technique known by the skilledperson including but not limited to Sanger dideoxy terminationsequencing, whole-genome sequencing, sequencing by hybridization,pyrosequencing, capillary electrophoresis, cycle sequencing, single-baseextension sequencing, solid-phase sequencing, high-throughputsequencing, massively parallel signature sequencing (MPSS), sequencingby reversible dye terminator, paired-end sequencing, near-termsequencing, exonuclease sequencing, sequencing by ligation, short-readsequencing, single-molecule sequencing, sequencing-by-synthesis,real-time sequencing, reverse-terminator sequencing, nanoporesequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD®sequencing, MS-PET sequencing, mass spectrometry, and combinationsthereof.

Optionally, DNA is fragmented randomly, generally by physical methods,prior to sequencing. The anellovirus DNA may be sequenced by anytechnique known in the art, including sequencing by ligation,pyrosequencing, sequencing-by-synthesis or single-molecule sequencing.Sequencing also includes PCR-based techniques, such as for examplequantitative PCR or emulsion PCR.

Sequencing is performed on the entire DNA contained in the biologicalsample, or on portions of the DNA contained in the biological sample. Itwill be immediately clear to the skilled person that the said samplecontains at least a mixture of anellovirus DNA and of DNA from the hostsubject. Moreover, the anellovirus DNA is likely to represent only aminor fraction of the total DNA present in the sample.

A first approach that addresses these challenges is to sequence andquantify sequences which are known to be specific of the anellovirusgenome. Indeed, the inventors have identified two consensus sequences,represented by SEQ ID No. 4 and SEQ ID No. 5, based on the comparisonbetween all the anelloviral sequences. These two consensus sequences,which are capable of hybridizing to all the anelloviral genomes, arethus highly convenient as primers for sequencing the said anellovirusDNA.

Thus according to this embodiment, the method of the invention comprisesusing the primers of sequences represented by SEQ ID No. 4 and SEQ IDNo. 5 for sequencing. In a preferred embodiment, the method of theinvention comprises a step of quantifying the number of reads.

In yet a further preferred embodiment, the method of the inventioncomprises another further step of normalizing the said number of readsto a reference. The said reference may be any convenient DNA sequencewhich can be identified and quantified, e.g. a host DNA sequence or anexogenous sequence. It is particularly advantageous for quantitativesequencing to add ab initio into the samples a known amount of referencenucleic acids; which reference nucleic acids will pass through allsample preparation steps before sequencing. Sample preparation steps cancomprise means to protect the viral nucleic acid and destroy hostnucleic acids, for example using different nucleases.

Although the said primers can be used in solution, it is preferable thatthe said primers are linked to a solid support.

To permit its covalent coupling to the support, the primer is generallyfunctionalized. Thus, it may be modified by a thiol, amine or carboxylterminal group at the 5′ or 3′ position. In particular, the addition ofa thiol, amine or carboxyl group makes it possible, for example, tocouple the oligonucleotide to a support bearing disulphide, maleimide,amine, carboxyl, ester, epoxide, cyanogen bromide or aldehyde functions.These couplings form by establishment of disulphide, thioether, ester,amide or amine links between the primer and the support. Any othermethod known to a person skilled in the art may be used, such asbifunctional coupling reagents, for example.

Moreover, to improve the hybridization with the coupled oligonucleotide,it can be advantageous for the oligonucleotide to contain an “arm” and a“spacer” sequence of bases. The use of an arm makes it possible, ineffect, to bind the primer at a chosen distance from the support,enabling its conditions of interaction with the DNA to be improved. Thearm advantageously consists of a linear carbon chain, comprising 1 to 18and preferably 6 or 12 (CH₂) groups, and an amine which permits bindingto the column. The arm is linked to a phosphate of the oligonucleotideor of a “spacer” composed of bases which do not interfere with thehybridization. Thus, the “spacer” can comprise purine bases. As anexample, the “spacer” can comprise the sequence GAGG. The arm isadvantageously composed of a linear carbon chain comprising 6 or 12carbon atoms.

For implementation of the present invention, different types of supportmay be used. These can be functionalized chromatographic supports, inbulk or prepacked in a column, functionalized plastic surfaces orfunctionalized latex beads, magnetic or otherwise. Chromatographicsupports are preferably used. As an example, the chromatographicsupports capable of being used are agarose, acrylamide or dextran aswell as their derivatives (such as Sephadex, Sepharose, Superose, etc.),polymers such as poly(styrene/divinylbenzene), or grafted or ungraftedsilica, for example. The chromatography columns can operate in thediffusion or perfusion mode.

Thus, in one particular embodiment, the above primers such as SEQ ID No4 and 5, or primers featuring at least 12, 15, 20 or 25 consecutivebases of SEQ ID No 4 or 5, or other primers specific for anellovirusnucleic acids amplification, further comprise:

-   -   a functional group for covalent coupling at the 5′ or 3′ end        such as but not limited to a group comprising a thiol, amine or        carboxyl terminal group;    -   a spacer molecule or sequence is added at the 5′ or 3′ end,        which spacer molecule or sequence is as featured above;    -   optionally, additional sequences as index or tag sequences to        perform pre or post additional and general amplification steps        not depending on the target sequences to be quantified.

Another approach is to use a method that allows the quantitativegenotyping of nucleic acids obtained from the biological sample withhigh precision. In one embodiment of this approach, the precision isachieved by analysis of a large number (for example, millions orbillions) of nucleic acid molecules without any amplification usingprotocols that relies on previous knowledge of the target sequences(i.e. in this case, anellovirus sequences). One embodiment usesmassively parallel DNA sequencing, such as, but not limited to thatperformed by the Illumina Genome Analyzer platform (Bentley et al.Nature; 456: 53-59, 2008), the Roche 454 platform (Margulies et al.Nature; 437: 376-380, 2005), the ABI SOLiD platform (McKernan et al.,Genome Res; 19: 1527-1541, 2009), the Helicos single molecule sequencingplatform (Harris et al. Science; 320: 106-109, 2008), real-timesequencing using single polymerase molecules (Science; 323: 133-138,2009), Ion Torrent sequencing (WO 2010/008480; Rothberg et al., Nature,475: 348-352, 2011) and nanopore sequencing (Clarke J et al. NatNanotechnol.; 4: 265-270, 2009). In one embodiment, massively parallelsequencing is performed on a random subset of nucleic acid molecules inthe biological sample.

In specific embodiments, the method and kit of the invention is adaptedto run on ABI PRISM® 377 DNA Sequencer, an ABI PRISM® 310, 3100,3100-Avant, 3730, or 3730×1 Genetic Analyzer, an ABI PRISM® 3700 DNAAnalyzer, or an Applied Biosystems SOLiD™ System (all from AppliedBiosystems), a Genome Sequencer 20 System (Roche Applied Science), anHiSeq 2500, an HiSeq 2000, a Genome Analyzer IIx, a MiSeq PersonalSequencer, a HiScanSQ (all from Illumina), the Genetic Analysis System,including the Single Molecule Sequencer, Analysis Engine and SampleLoader (all from HeliScope), the Ion Proton™ Sequencer, or the Ion PGM™Sequencer (both from Ion Torrent). In a preferred embodiment of theinvention, anellovirus sequences are identified in the global sequencingdata by comparison with the publicly-deposited anellovirus sequences.This comparison is advantageously based on the level of sequenceidentity with a known anellovirus sequence and allow to detect evendistant variants.

The term “sequence identity” refers to the identity between two peptidesor between two nucleic acids. Identity between sequences can bedetermined by comparing a position in each of the sequences which may bealigned for the purposes of comparison. When a position in the comparedsequences is occupied by the same base or amino acid, then the sequencesare identical at that position. A degree of sequence identity betweennucleic acid sequences is a function of the number of identicalnucleotides at positions shared by these sequences. A degree of identitybetween amino acid sequences is a function of the number of identicalamino acid sequences that are shared between these sequences. Since twopolypeptides may each (i) comprise a sequence (i.e. a portion of acomplete polynucleotide sequence) that is similar between twopolynucleotides, and (ii) may further comprise a sequence that isdivergent between two polynucleotides, sequence identity comparisonsbetween two or more polynucleotides over a “comparison window” refers tothe conceptual segment of at least 20 contiguous nucleotide positionswherein a polynucleotide sequence may be compared to a referencenucleotide sequence of at least 20 contiguous nucleotides and whereinthe portion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e. gaps) of 20 percent or lesscompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences.

To determine the percent identity of two amino acids sequences or twonucleic acid sequences, the sequences are aligned for optimalcomparison. For example, gaps can be introduced in the sequence of afirst amino acid sequence or a first nucleic acid sequence for optimalalignment with the second amino acid sequence or second nucleic acidsequence. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, the molecules are identical at that position. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences. Hence % identity=number ofidentical positions/total number of overlapping positions×100.

In this comparison the sequences can be the same length or can bedifferent in length. Optimal alignment of sequences for determining acomparison window may be conducted by the local homology algorithm ofSmith and Waterman (J. Theor. Biol., 91(2): 370-380, 1981), by thehomology alignment algorithm of Needleman and Wunsch (J. Mol. Biol,48(3): 443-453, 1972), by the search for similarity via the method ofPearson and Lipman (Proc. Natl. Acad. Sci. U.S.A., 85(5): 2444-2448,1988), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetic Computer Group, 575, Science Drive, Madison, Wis.)or by inspection. The best alignment (i.e. resulting in the highestpercentage of identity over the comparison window) generated by thevarious methods is selected.

The term “sequence identity” thus means that two polynucleotide orpolypeptide sequences are identical (i.e. on a nucleotide by nucleotideor an amino acid by amino acid basis) over the window of comparison. Theterm “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e. the window size)and multiplying the result by 100 to yield the percentage of sequenceidentity. The same process can be applied to polypeptide sequences. Thepercentage of sequence identity of a nucleic acid sequence or an aminoacid sequence can also be calculated using BLAST software (Version 2.06of September 1998) with the default or user defined parameter.

Thus, a sequence displaying at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identity with a known anellovirus sequence is identified as ananellovirus sequence. According to this embodiment, determining theanelloviral load thus includes numbering the anellovirus sequencesidentified by sequencing in the biological sample of the subject.

In a preferred embodiment, the levels of anellovirus DNA are determinedby measuring the number of anellovirus DNA sequences present in thebiological sample.

In a further preferred embodiment, the number of anelloviral sequencesis normalized to the total number of sequences identified by sequencingin the biological sample. By “normalizing to the total number ofsequences”, it is herein meant that the number of anelloviral sequencesis divided by the total number of sequences, i.e. both from anelloviraland non-anelloviral origin, present in the biological sample. Accordingto this embodiment, a ratio of anelloviral sequences to total sequenceshigher than 0.2×10⁻⁴ indicates that the subject is immunosuppressed. Ina yet further preferred embodiment, the subject is immunosuppressed ifthe said ratio is higher than 0.5×10⁻⁴, higher than 1×10⁻⁴, higher than5×10⁻⁴, higher than 10×10⁻⁴, higher than 15×10⁻⁴, higher than 20×10⁻⁴,higher than 25×10⁻⁴, or higher than 25×10⁻⁴.

In another embodiment, determining the anelloviral load further includesthe steps of assigning each anellovirus sequence identified bysequencing to a specific anellovirus genome and numbering the copies ofanellovirus genomes thus identified. By assigning a sequence to aspecific anellovirus genome, it is herein meant the process ofidentifying the anellovirus genome to which the said sequence belongs.Advantageously, this process is carried out by comparison of the saidsequence with the anelloviral sequences deposited in public databases.

By “anellovirus genomes”, it is herein referred to the genomes of anyvirus belonging to the Anelloviridae family of viruses, comprisingTorque Teno viruses (TTVs), Torque Teno midiviruses (TTMDVs), TorqueTeno miniviruses also formerly known as Torque Teno-like miniviruses(TTMVs).

In an embodiment, it is referred to TTVs genomes, comprisingalphatorqueviruses, betatorqueviruses, gammatorqueviruses,deltatorqueviruses, epsilontorqueviruses, etatorqueviruses,iotatorqueviruses, thetatorqueviruses, zetatorqueviruses. In a preferredembodiment, it is referred to the genome of the prototype strain ofTorque Teno virus, TTV-1a. An example of a TTV genome is a sequence suchas in e.g. Genebank accession number AB017610 and represented in SEQ IDNo 1.

In another embodiment, it is referred to TTMDVs genomes. In a preferredembodiment, it is referred to the genome of the prototype strain ofTorque Teno midiviruses, namely the genome of the MD1-073 isolate. Forexample, a TTMDV genome may have a sequence such as in e.g. Genbankaccession number AB290918 and represented in SEQ ID No 2.

In another embodiment, it is referred to TTMVs genomes. In a preferredembodiment, it is referred to the genome of the prototype strain ofTorque Teno miniviruses, namely the genome of TTMV-NLC-030 isolate. ATTMV genome is illustrated by a sequence such as in e.g. Genbankaccession number AB038631 and represented in SEQ ID No 3.

It is possible to normalize the number of anellovirus genomes to atleast one sequence, in order to reduce the error rate when comparing theanelloviral loads of two distinct biological samples. By “normalizing toa sequence”, it is herein meant that the number of anellovirus genomesidentified in the biological sample is divided by the number of copiesof the said sequence. The said sequence can be for example a non-humansequence whose copy number is known. Alternatively, the said sequence isa human sequence. Preferably, number of anellovirus genomes isnormalized to the whole human genome. By “human genome” it is hereinreferred to a consensus sequence of reference, such as the sequencecorresponding to Genome Reference Consortium built GRCh37 (NCBI build37.1/assembly hg19).

In a preferred embodiment, the ratio of anellovirus genomes to humangenome is higher than 0.2%.

Alternatively, the number of anelloviral sequences in a given sample iscompared to an internal control. The said internal control enables theassessment of the quality and the extent of the sequencing of thenucleic acid molecules in the said sample. Preferably, the said internalcontrol consists of a nucleic acid molecule having a known sequence,said nucleic acid molecule being present in the said sample at a knownconcentration. More preferably, the said nucleic acid molecule is thegenomic single-stranded circular DNA molecule of a virus of knownsequence and concentration in the said sample. Such a known virus may bee.g. a virus of the Circoviridae family. The ratio of the number ofsequences of the sample to the control allows estimating the absolutenumber of anelloviral genomes of known sequence and concentration.

In another embodiment, amplification techniques are used to determinethe anelloviral load. Such amplification techniques include inparticular isothermal methods and PCR-based techniques. Isothermaltechniques include such methods as e.g. nucleic acid sequence-basedamplification (NASBA), loop-mediated isothermal amplification (LAMP),helicase-dependent amplification (HDA), rolling circle amplification(RCA), and strand displacement amplification (SDA), exponentialamplification reaction (EXPAR), isothermal and chimeric primer-initiatedamplification of nucleic acids (ICANs), signal-mediated amplification ofRNA technology (SMART) and others (see e.g. Asiello and Baeumner, LabChip; 11(8): 1420-1430, 2011). Preferably, the PCR technique usedquantitatively measures starting amounts of DNA, cDNA, or RNA. Examplesof PCR-based techniques according to the invention include techniquessuch as, but not limited to, quantitative PCR (Q-PCR),reverse-transcriptase polymerase chain reaction (RT-PCR), quantitativereverse-transcriptase PCR (QRT-PCR), or digital PCR. These techniquesare well known and easily available technologies for those skilled inthe art and do not need a precise description.

In a preferred embodiment, the determination of anelloviral load isperformed by quantitative PCR.

In another preferred embodiment, the determination of the adenoviralload is performed by digital PCR. Digital PCR involves multiple PCRanalyses on extremely dilute nucleic acids such that most positiveamplifications reflect the signal from a single template molecule.Digital PCR thereby permits the counting of individual templatemolecules. The proportion of positive amplifications among the totalnumber of PCRs analyzed allows an estimation of the templateconcentration in the original or non-diluted sample. This technique hasbeen proposed to allow the detection of a variety of genetic phenomena(Vogelstein et al., Proc Natl Acad Sci USA 96: 9236-924, 1999). Sincetemplate molecule quantification by digital PCR does not rely ondose-response relationships between reporter dyes and nucleic acidconcentrations, its analytical precision is, at least theoretically,superior to that of real-time PCR. Hence, digital PCR potentially allowsthe discrimination of finer degrees of quantitative differences betweentarget and reference loci.

In another embodiment, the method of the invention comprises a furtherstep (a′) of comparing the anelloviral load of step (a) to at least onereference anelloviral load. According to this embodiment, thedetermination of the immunosuppressed or non-immunosuppressed phenotypeis carried out thanks to the obtained anelloviral load with at least onereference anelloviral load in step (a′).

According to the invention, the “reference anelloviral load” is apredetermined measure of anelloviral load, obtained from a biologicalsample from a subject with a known immune status. Preferably, thereference anelloviral load is predetermined measure of anelloviral load,obtained from a biological sample from a subject with a knownimmunosuppressed status. In a preferred embodiment, the referenceanelloviral load is predetermined measure of anelloviral load, obtainedfrom a biological sample from a subject known to be immunosuppressed. Inanother embodiment, the reference anelloviral load is predeterminedmeasure of anelloviral load, obtained from a biological sample from asubject known not to be immunosuppressed.

Preferably, at least one reference expression profile is animmunosuppressed reference anelloviral load. Alternatively, at least onereference anelloviral load may be a non-immunosuppressed referenceanelloviral load. More preferably, the determination of the presence orabsence of an immunosuppressed phenotype is carried out by comparisonwith at least one immunosuppressed and at least one non-immunosuppressedreference anelloviral load. The diagnosis or prognostic may thus beperformed using one immunosuppressed reference anelloviral load and onenon-immunosuppressed reference anelloviral load. Advantageously, to geta stronger diagnosis or prognostic, said diagnosis or prognostic iscarried out using several immunosuppressed reference anelloviral loadsand several non-immunosuppressed reference anelloviral loads.

According to the invention, any sequence of the anellovirus genome maybe targeted for amplification in order to determine the anelloviralload. The said sequence may be conserved amongst the viral strains or itmay be only present in a specific group of strains. In the latter case,it is necessary to amplify as well another sequence present in the othergroups of anellovirus strains. Preferably, the amplified anellovirussequence is conserved among the Anelloviridiae family, in which case asingle set of primers may be used to amplify, in a single reaction, allthe viral strains present in the biological sample. More preferably,more than one conserved sequences in the Anelloviridiae family areamplified, thus increasing the sensitivity of the test.

In a specific embodiment, a reference sequence is also targeted foramplification, and used as a control or as a standard. Preferably, thisreference sequence is chosen within the human genome.

In a preferred embodiment, the determination of anelloviral load isperformed using Q-PCR; at least one consensus sequence of anellovirusgenome is targeted for amplification.

The primers are chosen by the skilled in the art depending on thedesired specificity of the PCR amplification step using standardparameters such as the nucleic acid size, GC contents, and temperaturereactions.

In a more preferred embodiment, any anelloviral sequence comprisedbetween the consensus sequences represented by SEQ ID No. 4 and SEQ IDNo. 5 is targeted for amplification. Even more preferably, theanelloviral sequence comprised between the consensus sequencesrepresented by SEQ ID No. 4 and SEQ ID No. 5 is targeted foramplification.

The present inventors have identified two consensus sequences,represented by SEQ ID No. 4 and SEQ ID No. 5, based on the comparisonbetween all the anelloviral sequences. Preferably, when amplification isperformed, amplification primers comprise any primers capable of bindingto a polynucleotide having said consensus sequences SEQ ID No.4 or SEQID No.5, with the proviso that the said primers are different from SEQID No.6 and SEQ ID No.7. In a specific embodiment, the said primerscomprise between 10 and 30 nucleotides, preferably between 15 and 25nucleotides, more preferably between 20 and 25 nucleotides. Morepreferably, the said primers comprise at least 12, 15, 20 or 25 bases ofSEQ ID No. 4 or 5. Parameters for determining the exact primer sequenceon the basis of the target sequence are well known to the person ofskills in the art.

Although the said primers can be used in solution, it is preferable thatthe said primers are linked to a solid support.

To permit its covalent coupling to the support, the primer is generallyfunctionalized. Thus, it may be modified by a thiol, amine or carboxylterminal group at the 5′ or 3′ position. In particular, the addition ofa thiol, amine or carboxyl group makes it possible, for example, tocouple the oligonucleotide to a support bearing disulphide, maleimide,amine, carboxyl, ester, epoxide, cyanogen bromide or aldehyde functions.These couplings form by establishment of disulphide, thioether, ester,amide or amine links between the primer and the support. Any othermethod known to a person skilled in the art may be used, such asbifunctional coupling reagents, for example.

Moreover, to improve the hybridization with the coupled oligonucleotide,it can be advantageous for the oligonucleotide to contain an “arm” and a“spacer” sequence of bases. The use of an arm makes it possible, ineffect, to bind the primer at a chosen distance from the support,enabling its conditions of interaction with the DNA to be improved. Thearm advantageously consists of a linear carbon chain, comprising 1 to 18and preferably 6 or 12 (CH₂) groups, and an amine which permits bindingto the column. The arm is linked to a phosphate of the oligonucleotideor of a “spacer” composed of bases which do not interfere with thehybridization. Thus, the “spacer” can comprise purine bases. As anexample, the “spacer” can comprise the sequence GAGG. The arm isadvantageously composed of a linear carbon chain comprising 6 or 12carbon atoms.

For implementation of the present invention, different types of supportmay be used. These can be functionalized chromatographic supports, inbulk or prepacked in a column, functionalized plastic surfaces orfunctionalized latex beads, magnetic or otherwise. Chromatographicsupports are preferably used. As an example, the chromatographicsupports capable of being used are agarose, acrylamide or dextran aswell as their derivatives (such as Sephadex, Sepharose, Superose, etc.),polymers such as poly(styrene/divinylbenzene), or grafted or ungraftedsilica, for example. The chromatography columns can operate in thediffusion or perfusion mode.

In another embodiment of the invention, the quantity of anellovirus DNAis determined using sequence specific hybridization. The terms“hybridization” and “hybridizing” refers to the pairing of twocomplementary single-stranded nucleic acid molecules (RNA and/or DNA) togive a double-stranded molecule. As used herein, two nucleic acidmolecules may be hybridized, although the base pairing is not completelycomplementary. Accordingly, mismatched bases do not preventhybridization of two nucleic acid molecules provided that appropriateconditions, well known in the art, are used.

The probes are chosen by the person skilled in the art depending on thedesired specificity of the specificity of the detection step usingstandard parameters such as the nucleic acid size and GC contents,stringent hybridization conditions and temperature reactions. Forexample, low stringency conditions are used when it is desired to obtainbroad positive results on a range of homologous targets whereas highstringency conditions are preferred to obtain positive results only ifthe specific target nucleic is present in the sample.

Preferably, the probe of the invention is capable of hybridizing with apolynucleotide having a sequence represented by SEQ ID No.4 or SEQ IDNo. 5, with the proviso that the said probe does not have a sequencerepresented by SEQ ID No. 6 or SEQ ID No. 7. It is another aspect of theinvention to provide a polynucleotide comprising at least 12, 15, 20 orconsecutive bases represented by SEQ ID No. 4 or SEQ ID No. 5.

The hybridizing probes may be labeled with a radioactive marker, afluorescent marker, or a chemical marker. Branched DNA (bDNA), involvesoligonucleotides with branched structures that allow each individualoligonucleotide to carry 35 to 40 labels (e.g., alkaline phosphataseenzymes). These procedures include, but are not limited to, DNA-DNA orDNA-RNA hybridizations, PCR amplification, and protein bioassay orimmunoassay techniques which include membrane, solution, or chip basedtechnologies for the detection and/or quantification of nucleic acid orprotein sequences. Such methods of hybridization of a labeled probe witha DNA molecule are well known to the person of skills in the art. Forexample, low stringency conditions are used when it is desired to obtainbroad positive results on a range of homologous targets whereas highstringency conditions are preferred to obtain positive results only ifthe specific target nucleic is present in the sample. As used herein,the term “stringent hybridization conditions” refers to conditions underwhich the probe will hybridize only to that exactly complementary targetsequence, and which allow the detection of the specific target sequence.The hybridization conditions affect the stability of hybrids, e.g.,temperature, salt concentration, pH, formamide concentration and thelike. These conditions are optimized to maximize specific binding andminimize non-specific binding of primer or probe to its target nucleicacid sequence. Stringent conditions are sequence dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequences at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe orprimer. Typically, stringent conditions will be those in which the saltconcentration is less than about 1.0 M Na⁺, typically about 0.01 to 1.0M Na⁺ concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes or primers (e.g.10 to 50 nucleotides) and at least about 60° C. for long probes orprimers (e.g. greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. Exemplary low stringent conditions include hybridization witha buffer solution of 20-30% formamide, 1 M NaCl, 1% SDS at 37° C. and awash in 2×SSC at 40° C. Exemplary high stringency conditions includehybridization in 40-50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1*SSC at 60° C. Determination of particular hybridizationconditions relating to a specified nucleic acid is routine and is wellknown in the art, for instance, as described in Sambrook and Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; 3rd Ed., 2001; and Ausubel, Ed., Short Protocols in MolecularBiology, Current Protocols; 5th Ed., 2002.

In a preferred embodiment, the anelloviral load is determined by the useof a nucleic microarray.

According to the invention, a “nucleic microarray” consists of differentnucleic acid probes that are attached to a substrate, which can be amicrochip, a glass slide or a microsphere-sized bead. A microchip may beconstituted of polymers, plastics, resins, polysaccharides, silica orsilica-based materials, carbon, metals, inorganic glasses, ornitrocellulose. Probes can be nucleic acids such as cDNAs (“cDNAmicroarray”) or oligonucleotides (“oligonucleotide microarray”), and theoligonucleotides may be about 25 to about 60 base pairs or less inlength.

To determine the amount of a target nucleic sample, said sample islabelled, contacted with the microarray in hybridization conditions,leading to the formation of complexes between target nucleic acids thatare complementary to probe sequences attached to the microarray surface.The presence of labelled hybridized complexes is then detected. Manyvariants of the microarray hybridization technology are available to theman skilled in the art.

The practice of the invention employs, unless other otherwise indicated,conventional techniques or protein chemistry, molecular virology,microbiology, recombinant DNA technology, and pharmacology, which arewithin the skill of the art. Such techniques are explained fully in theliterature. (See Ausubel et al., Short Protocols in Molecular Biology,Current Protocols; 5th Ed., 2002; Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Co., Easton, Pa., 1985; and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; 3rd Ed., 2001). The nomenclatures used in connection with, andthe laboratory procedures and techniques of, molecular and cellularbiology, protein biochemistry, enzymology and medicinal andpharmaceutical chemistry described herein are those well-known andcommonly used in the art.

The following examples are provided herein for purposes of illustrationonly and are not intended to be limiting unless otherwise specified.

EXAMPLES Example 1

ANV as a Marker of Immune Suppression

We have screened plasma samples from patients suffering from variousforms of congenital immune suppression and from patients without anyknown immunosuppression: the extraction procedure was optimized forisolation of viral DNA or RNA genomes without precluding identificationof bacterial and fungal nucleic acids. A volume of 150 μl of each samplewas extracted using the Nucleospin RNA virus kit (Macherey-Nagel), whichallows recovery of both DNA and RNA, and then amplified by thebacteriophage phi29 polymerase based multiple displacement amplification(MDA) assay using random primers.

This technique allows DNA synthesis from DNA samples, and also from cDNAfragments from viral genomes previously colligated prior to Phi29polymerase-MDA.

Briefly, the protocol of the QuantiTect Whole Transcriptome Kit (Qiagen)was followed, except that the cDNA synthesis step was performed withrandom hexamer primers.

-   -   A mix with 8 μl of RNA, 1 μl of primer (50 μM) and 1 μl of dNTPs        (10 mM) was incubated at 75° C. for 5 min and cooled on ice for        5 min.    -   Then, 10 μl of 2× enzyme mix were added. This enzyme mix was        composed of 2 μl of 10× RT Buffer for SSIII (Invitrogen Inc.), 4        μl of 25 mM MgCl₂, 2 μl μL of 0.1 M DTT, 1 μl μL of 40 U/μl        RNaseOUT (Invitrogen Inc.), 1 μl of SuperScript III reverse        transcriptase (Invitrogen Inc.) and 0.5 μl of DMSO        (Sigma-Aldrich).    -   The final mix was incubated at 25° C. for 10 min, then at 45° C.        for 90 min and finally at 95° C. for 5 min.    -   All cDNAs were stored at −20° C. or immediately used.    -   The two following steps (ligation and WGA) were performed with        the QuantiTect® Whole Transcriptome kit (Qiagen) according to        the manufacturer's instructions.

This provides concatemers of high molecular weight DNA.

High molecular weight DNA (5 μg), resulting from isothermalamplification of the pool of genomic DNAs and cDNAs made from genomicRNAs as described above, was fragmented into 200 to 350 nt fragments, towhich adapters were ligated. Adapters included a nucleotide tag allowingfor multiplexing several samples per lane or channel. Sequencing wasconducted on an Illumina GAII or HiSeq 2000 sequencer.

Sorting out the flow of Illumina sequences was first done by asubtractive database comparison procedure. To this end, the whole humangenome sequence (NCBI build 37.1 /assembly hg19) was scanned with thereads using SOAPaligner. A quick and very restrictive BLASTN study wasalso performed to eliminate additional host reads. The best parametersto be used have been determined previously. A number of assemblyprograms dedicated to short or medium-sized reads (Velvet, SOAPdenovo,CLC) have been tested for their efficiency in our pipeline. Optimalparameters have been set. The comparison of the single reads and contigswith already known genomic and taxonomic data was done on dedicatedspecialized viral, bacterial and generalist databases maintained locally(GenBank viral and bacterial databases, nr). The aforementioneddatabases were scanned using BLASTN and BLASTX. Binning (or taxonomicassignment) was based on the lowest common ancestor or from the besthit, identified from the best hits among single reads or contigs with asignificant e-value.

Results regarding single reads with e-values below 10⁻⁴ that had a bestmatch for a known member of the Anelloviridae family (ANV) are shown intable 1.

As shown in table 1, we have identified ANV reads in 14/14immunodeficient patients. Interestingly, for a subset of three patientscharacterized with a major immune T-cell suppression, 20 to 54% of thetotal number of reads were anellovirus reads. On the contrary, otherimmune compromised patients that showed lower immunosuppression statesdepicted<0.2% anellovirus virus reads.

In 34 patients not known to be immune-compromised, no read (or less than5 reads) was identified except in a set of four patients: three of them(100036, 100039, 080114/116) were developing an encephalitis at the timeof sampling, a medical condition frequently associated with immunesuppression.

TABLE 1 Number of single reads or of reads assembled in contigsidentified for patients with immunosuppression or without knownimmunosuppression. ratio Immunosuppression number of anellovirusanello/total Code Sample state reads reads (×10⁻⁴) KNOWNIMMUNOSUPPRESSSION 100023 Plasma SCID gamma 5 132 262 191  0.37C/hepatitis 100025 Plasma SCID/HPV 5 345 602 2 889 856      5406.04infections 100026 Plasma Bruton's 3 091 732 94  0.30 Agammaglobuli nemia100028 Plasma SCID gamma 3 506 376 13  0.04 C/encephalopathy 100029Plasma Severe 6 467 110 3 299 612      5102.14 lymphopenia AtypicalStill 100030 Plasma disease/fever 12 029 412  13  0.01 of unknown origin100031 Plasma Septic 2 770 398 2 871    10.36 granulomatosis 100061Plasma Severe 9 184 316 526  0.57 lymphopenia 100081 Plasma Griscelli 8540 162 5 272    6.17 disease and CMV infection 100082 CerebrospinalSCID Rag1 20 875 934  1 805    0.86 Fluid 100083 Plasma Severe 9 643 09616 129    16.73 lymphopenia 100084 Plasma Severe 8 128 858 1 705998      2098.69 lymphopenia 100085 Plasma leukemia 6 410 912 286  0.45100243 Skin swab Severe 39 942 410  132 761     33.24 lymphopeniaCONTROLS 100036 Plasma NIL 13 564 262  187  0.14 100039 Plasma NIL 6 071374 915  1.51 080114/ Plasma NIL 9 633 342 164  0.17 116 100053 PlasmaNIL 7 375 016 0 0.00 100032 Plasma NIL 7 148 184 0 0.00 100033 PlasmaNIL 3 348 242 4 0.01 100034 Plasma NIL 14 198 534  0 0.00 100035 PlasmaNIL 13 905 700  0 0.00 100037 Plasma NIL 13 784 866  0 0.00 100038Plasma NIL 27 941 897  0 0.00 100062 Plasma NIL 12 098 932  2 0.00100063 Plasma NIL 8 701 704 1 0.00 100064 Plasma NIL 7 976 148 0 0.00100066 Plasma NIL 8 052 770 156  0.19 100067 Plasma NIL 10 354 496  20.00 100069 Plasma NIL 9 107 144 0 0.00 100070 Plasma NIL 8 196 240 20.00 100072 Plasma NIL 7 588 712 0 0.00 100073 Plasma NIL 10 281 130  00.00 100192 Plasma NIL 28 726 064  0 0.00 100236 Plasma NIL 4 818 410 00.00 100237 Plasma NIL 6 047 999 0 0.00 100238 Plasma NIL 4 742 180 00.00 100239 Plasma NIL 4 837 188 0 0.00 100240 Plasma NIL 5 281 443 00.00 100241 Plasma NIL 9 226 049 0 0.00

Example 2

Identification of Conserved Sequences Among ANVs Genomes

An ANV sequence database was built. It was composed by 7,003 nucleotidesequences assigned to the Anelloviridae family. 6938 sequences come fromthe NCBI public database (NT, Oct. 1, 2011). 65 sequences come from denovo assembly of NGS reads derived from our own work (see table 1) thatwere assigned to the Anelloviridae family. These sequences have aminimal length of 1000 nucleotides.

In a preliminary analysis using a short set of sequences, sequences thatare close to a transcriptional factor binding site (called TATA box)seemed conserved among strains. To further extend this analysis, and inorder to reduce the database size, filter based on the presence of theTATA box was performed. This motif is A/T rich and was located upstreamof the transcription start site.

A consensus sequence of the TATA box was built, using the followingsequences: ATAAAA, ATAAAT, ATATAA, ATATAT.

This motif was used as an anchor in order to:

-   -   1. Filter the anellovirus database. A search with a position        specific weight matrix was performed with this consensus.        Sequence without an exact match was discarded.    -   2. Pre-aligned long nucleotide sequences. Previously selected        sequences were aligned at the beginning of the TATA consensus        (+1).

This filtering step reduced the database to:

-   -   42 sequences obtained from the NCBI.    -   8 sequences obtained from our own NGS runs and de novo assembly.

A second alignment was done with MUSCLE (default parameters). Twoconserved regions were identified at position [+20: +53] and position[+121: +151] relative to the TATA box consensus.

Details of these alignments are shown below. Each line corresponds to aspecific nucleotide position. Columns represent respectively the A, C, Gand T counts at this position. Lines with a star correspond to awell-conserved position. Nucleotide ambiguity was shown using bracket.The last column is the consensus nucleotide at each position using theIUAPAC nomenclature.

TABLE 2 IUAPAC Nomenclature Nucleotide Code Base A Adenine C Cytosine GGuanine T (or U) Thymine (or Uracil) R A or G Y C orT S G or C W A orT KG orT M A or C B C or G or T D A or G or T H A or C or T V A or C or G Nany base or - gap

A C G T First region: ANV-PTQ-1 0 0 50 0 * G G 50 0 0 0 * A A 50 0 0 0 *A A 0 0 0 50 * T T 0 0 50 0 * G G 0 0 50 0 * G G 0 37 0 13 [CT] Y 13 0 037 [AT] W 0 0 50 0 * G G 50 0 0 0 * A A 0 0 50 0 * G G 0 0 0 50 * T T 00 0 50 * T T 0 0 0 50 * T T 0 2 0 11 [CT] Y 14 0 0 36 [AT] W 0 36 0 14[CT] C 0 24 14 12 [CGT] B 23 14 0 13 [ACT] H 0 49 1 0 C C 0 0 50 0 * G G0 50 0 0 * C C 10 37 0 3 [AC] C 2 35 12 1 [CG] S 0 0 50 0 * G G 4 0 0 36[AT] T 0 38 0 12 [CT] Y 0 36 14 0 [CG] S 0 0 50 0 * G G 0 36 0 12 [CT] Y36 0 14 0 [AG] R 0 0 50 0 * G G 18 1 0 0 [AT] A Second region: ANV-PTQ-250 0 0 0 * A A 0 38 12 0 [CG] S 0 50 0 0 * C C 0 0 50 0 * G G 16 21 12 1[ACG] V 50 0 0 0 * A A 0 0 50 0 * G G 0 0 0 50 * T T 0 50 0 0 * C C 49 00 1 * A A 50 0 0 0 * A A 0 0 50 0 * G G 0 0 50 0 * G G 0 0 50 0 * G G 00 50 0 * G G 0 50 0 0 * C C 38 10 0 2 [AC] M 38 0 0 12 [AT] W 12 0 0 38[AT] W 0 0 0 50 * T T 0 50 0 0 * C C 0 0 50 0 * G G 0 0 50 0 * G G 0 050 0 * G G 0 50 0 0 * C C 16 0 10 24 [AGT] D 2 36 12 0 [CG] S 0 0 50 0 *G G 1 12 37 0 [CG] S 0 0 10 4 [GT] K 1 12 37 0 [CG] S 0 0 10 4 [GT] G 02 12 0 G G 2 2 0 10 T T 12 0 37 0 [AG] R 46 0 0 3 A A

The present inventors have thus identified the two following consensussequences which are conserved among anelloviral strains.

ANV-PTQ-2 (SEQ ID No. 4) 5′ ASCGVAGTCAAGGGGCMWWTCGGGCDSGSKSGGTRA 3′ANV-PTQ-1 (SEQ ID No. 5) 5′ GAATGGYWGAGTTTYWCBHCGCCSGTYSGYRGA 3′

Thus, primers can be defined within the following sequences and be usedto develop PCRs so as to evidence a wide range of ANVs genome.

Example 3

At least 50 and up to 200 immunocompromized patients from hospitalNecker (Paris) suffering from an infectious disease are enrolled.Immunosuppression in these patients is either due to a congenitaldefect, to immunosuppressive treatments post solid organ or bone marrowtransplantation, or to evolution of a cancer.

High throughput sequencing is conducted on the plasma of each patient toevaluate the etiology of the disease and the anellovirus load as apotential marker of immunosuppression. The number of reads specific toanelloviruses is quantified and correlated with the immunosuppressionlevel of each patient and with the progression of the disease.

Non immunocompromized patients serve as controls.

It is observed that high loads in anellovirus in patients correlate withincreased immunosuppression.

The invention claimed is:
 1. A method of stimulating the immune systemof a human subject in need thereof, comprising 1) obtaining a bloodsample from the human subject, 2) measuring the level of anellovirus DNAin the sample by a method comprising detecting Torque teno virus (TTV)DNA, Torque teno midi virus (TTMDV) DNA, and Torque teno mini virus(TTMV) DNA, 3) calculating the ratio of anelloviral sequences to thetotal number of sequences present in the sample, wherein said measuredratio is higher than 0.2×10⁻⁴, and 4) administering a treatment tostimulate the immune system of the human subject.
 2. The method of claim1, wherein said human subject suffers from transplant rejection.
 3. Themethod of claim 1, wherein said human subject suffers from an infection.4. A method of immunosuppressing the immune system of a human subject inneed thereof, comprising 1) obtaining a blood sample from the humansubject, 2) measuring the level of anellovirus DNA in the sample by amethod comprising detecting Torque teno virus (TTV) DNA, Torque tenomidi virus (TTMDV) DNA, and Torque teno mini virus (TTMV) DNA, 3)calculating the ratio of anelloviral sequences to the total number ofsequences present in the sample, wherein said measured ratio is lowerthan 0.2×10⁻⁴, and 4) administering a treatment to suppress the immunesystem of the human subject.
 5. The method of claim 4, wherein saidhuman subject suffers from transplant rejection.
 6. The method of claim4, wherein said human subject suffers from an infection.
 7. The methodof claim 1, wherein the level of anellovirus DNA is measured byhybridization with a labeled probe, PCR amplification or sequencing. 8.The method of claim 1, wherein the level of anellovirus DNA is measuredby sequencing said anellovirus DNA.
 9. The method of claim 1, whereinthe level of anellovirus DNA is measured by a quantitative sequencingmethod comprising at least one amplification step with primerscomprising at least 12, 15, 20 or 25 consecutive bases of SEQ ID NO: 4or
 5. 10. The method of claim 9 wherein said primers further comprise atleast one of: a functional group for covalent coupling at the 5′ or 3′end, a spacer molecule or sequence at the 5′ or 3′ end, additionalsequences as index or tag sequences to perform pre or post additionaland general amplification steps not depending on the target sequences tobe quantified.
 11. The method of claim 1, wherein the level ofanellovirus DNA is measured by massive parallel sequencing.
 12. Themethod of claim 1, wherein measuring the level of anellovirus DNA in thesample comprises the step of measuring the number of anelloviralsequences in the said biological sample.
 13. The method of claim 1,wherein measuring the level of anellovirus DNA in the sample furthercomprises the steps of assigning each anellovirus sequence to a specificanellovirus genome and numbering the copies of anellovirus genomes thusidentified.
 14. The method of claim 13, wherein measuring the level ofanellovirus DNA in the sample further comprises the step of normalizingthe number of anellovirus genomes to at least one human or non-humansequence.
 15. The method of claim 1, wherein the level of anellovirusDNA in the sample is determined by quantitative PCR.
 16. The method ofclaim 1, wherein measuring the level of anellovirus DNA in the samplecomprises the amplification of at least one consensus anellovirussequence.
 17. The method of claim 1, wherein measuring the level ofanellovirus DNA in the sample comprises the amplification of anyanelloviral sequence that is between the consensus sequences of SEQ IDNo. 4 and SEQ ID No.
 5. 18. The method of claim 10 wherein saidfunctional group for covalent coupling at the 5′ or 3′ end is a terminalgroup comprising a thiol, amine or carboxyl group.
 19. The method ofclaim 4, wherein the level of anellovirus DNA is measured byhybridization with a labeled probe, PCR amplification or sequencing. 20.The method of claim 4, wherein the level of anellovirus DNA is measuredby sequencing said anellovirus DNA.
 21. The method of claim 4, whereinthe level of anellovirus DNA is measured by a quantitative sequencingmethod comprising at least one amplification step with primerscomprising at least 12, 15, 20 or 25 consecutive bases of SEQ ID NO: 4or
 5. 22. The method of claim 21 wherein said primers further compriseat least one of: a functional group for covalent coupling at the 5′ or3′ end, a spacer molecule or sequence at the 5′ or 3′ end, additionalsequences as index or tag sequences to perform pre or post additionaland general amplification steps not depending on the target sequences tobe quantified.
 23. The method of claim 4, wherein the level ofanellovirus DNA is measured by massive parallel sequencing.
 24. Themethod of claim 4, wherein measuring the level of anellovirus DNA in thesample comprises the step of measuring the number of anelloviralsequences in the said biological sample.
 25. The method of claim 4,wherein measuring the level of anellovirus DNA in the sample furthercomprises the steps of assigning each anellovirus sequence to a specificanellovirus genome and numbering the copies of anellovirus genomes thusidentified.
 26. The method of claim 25, wherein measuring the level ofanellovirus DNA in the sample further comprises the step of normalizingthe number of anellovirus genomes to at least one human or non-humansequence.
 27. The method of claim 4, wherein the level of anellovirusDNA in the sample is determined by quantitative PCR.
 28. The method ofclaim 4, wherein measuring the level of anellovirus DNA in the samplecomprises the amplification of at least one consensus anellovirussequence.
 29. The method of claim 4, wherein measuring the level ofanellovirus DNA in the sample comprises the amplification of anyanelloviral sequence that is between the consensus sequences of SEQ IDNo. 4 and SEQ ID No.
 5. 30. The method of claim 22 wherein saidfunctional group for covalent coupling at the 5′ or 3′ end is a terminalgroup comprising a thiol, amine or carboxyl group.
 31. A methodcomprising 1) obtaining a blood sample from a human subject undergoingan immunomodulation treatment, 2) measuring the level of anellovirus DNAin the sample by a method comprising detecting Torque teno virus (TTV)DNA, Torque teno midi virus (TTMDV) DNA, and Torque teno mini virus(TTMV) DNA, 3) calculating the ratio of anelloviral sequences to thetotal number of sequences present in the sample, wherein said measuredratio is about 0.2×10⁻⁴, and 4) continuing the immunomodulationtreatment.
 32. The method of claim 31, wherein said human subjectsuffers from transplant rejection.
 33. The method of claim 31, whereinsaid human subject suffers from an infection.
 34. The method of claim31, wherein the level of anellovirus DNA is measured by hybridizationwith a labeled probe, PCR amplification or sequencing.
 35. The method ofclaim 31, wherein the level of anellovirus DNA is measured by sequencingsaid anellovirus DNA.
 36. The method of claim 31, wherein the level ofanellovirus DNA is measured by a quantitative sequencing methodcomprising at least one amplification step with primers comprising atleast 12, 15, 20 or 25 consecutive bases of SEQ ID NO: 4 or
 5. 37. Themethod of claim 36 wherein said primers further comprise at least oneof: a functional group for covalent coupling at the 5′ or 3′ end, aspacer molecule or sequence at the 5′ or 3′ end, additional sequences asindex or tag sequences to perform pre or post additional and generalamplification steps not depending on the target sequences to bequantified.
 38. The method of claim 31, wherein the level of anellovirusDNA is measured by massive parallel sequencing.
 39. The method of claim31, wherein measuring the level of anellovirus DNA in the samplecomprises the step of measuring the number of anelloviral sequences inthe said biological sample.
 40. The method of claim 31, whereinmeasuring the level of anellovirus DNA in the sample further comprisesthe steps of assigning each anellovirus sequence to a specificanellovirus genome and numbering the copies of anellovirus genomes thusidentified.
 41. The method of claim 40, wherein measuring the level ofanellovirus DNA in the sample further comprises the step of normalizingthe number of anellovirus genomes to at least one human or non-humansequence.
 42. The method of claim 31, wherein the level of anellovirusDNA in the sample is determined by quantitative PCR.
 43. The method ofclaim 31, wherein measuring the level of anellovirus DNA in the samplecomprises the amplification of at least one consensus anellovirussequence.
 44. The method of claim 31, wherein measuring the level ofanellovirus DNA in the sample comprises the amplification of anyanelloviral sequence that is between the consensus sequences of SEQ IDNo. 4 and SEQ ID No.
 5. 45. The method of claim 37 wherein saidfunctional group for covalent coupling at the 5′ or 3′ end is a terminalgroup comprising a thiol, amine or carboxyl group.